Stroke, defined as a sudden weakening or loss of consciousness, sensation and volontary motion caused by rupture or obstruction of an artery of the brain, is the third cause of death in the United States. Worldwide, stroke is the number one cause of death due to its particularly high incidence in Asia. Ischemic stroke is the most common form of stroke, being responsible for about 85% of all strokes, whereas hemorrhagic strokes (e.g. intraparenchymal or subarachnoid) account for the remaining 15%. Due to the increasing mean age of the population, the number of strokes is continuously increasing. Because the brain is highly vulnerable to even brief ischemia and recovers poorly, primary prevention in ischemic stroke prevention offers the greatest potential for reducing the incidence of this disease.
Focal ischemic cerebral infarction occurs when the arterial blood flow to a specific region of the brain is reduced below a critical level. Cerebral artery occlusion produces a central acute infarct and surrounding regions of incomplete ischemia (sometimes referred to as ‘penumbra’), that are dysfunctional—yet potentially salvageable. Ischemia of the myocardium, as a result of reduced perfusion due to chronic narrowing of blood vessels, may lead to fatal heart failure and constitutes a major health threat. Acute myocardial infarction, triggered by coronary artery occlusion, produces cell necrosis over a time period of several hours. In the absence of reflow or sufficient perfusion, the cerebral or myocardial ischemic regions undergo progressive metabolic deterioration, culminating in infarction, whereas restoration of perfusion in the penumbra of the brain infarct or in the jeopardized but salvageable region of the myocardium may ameliorate the tissue damage.
Growth factor mediated improved perfusion of the penumbra in the brain or of the jeopardized myocardium of patients suffering ischemic events, either via increased vasodilation or angiogenesis (the formation of endothelial-lined vessels), may be of great therapeutic value according to Isner et al. in J. Clin. Invest. (1999) 103(9):1231-6 but many questions yet remain to be answered in this respect, for instance which suitable growth factor or combination of growth factors should be selected and which route of administration is effective yet safe for this purpose. In addition, an outstanding question is whether formation of new endothelial-lined vessels (i.e. angiogenesis) alone is sufficient to stimulate sustainable functional tissue perfusion. Indeed, coverage of endothelial-lined vessels by vascular smooth muscle cells (i.e. arteriogenesis) provides vasomotor control, structural strength and integrity and renders new vessels resistant to regression.
Capillary blood vessels consist of endothelial cells and pericytes, which carry all the genetic information required to form tubes, branches and entire capillary networks. Specific angiogenic molecules can initiate this process. A number of polypeptides which stimulate angiogenesis have been purified and characterized as to their molecular, biochemical and biological properties, as reviewed by Klagsbrun et al. in Ann. Rev. Physiol. (1991) 53:217-239 and by Folkman et al. in J. Biol. Chem. (1992) 267:10931-4. One factor that can stimulate angiogenesis and which is highly specific as a mitogen for vascular endothelial cells, is termed vascular endothelial growth factor (hereinafter referred as VEGF) according to Ferrara et al. in J. Cell. Biochem. (1991) 47:211-218. VEGF is also known as vasculotropin. Connolly et al. also describe in J. Biol. Chem. (1989) 264:20017-20024, in J. Clin. Invest. (1989) 84:1470-8 and in J. Cell. Biochem. (1991) 47:219-223 a human vascular permeability factor that stimulates vascular endothelial cells to divide in vitro and promotes the growth of new blood vessels when administered into healing rabbit bone grafts or rat corneas. The term vascular permeability factor (VPF for abbreviation) was adopted because of increased fluid leakage from blood vessels following intradermal injection and appears to designate the same substance as VEGF. The murine VEGF gene has been characterized and its expression pattern in embryogenesis has been analyzed. A persistent expression of VEGF was observed in epithelial cells adjacent to fenestrated endothelium, e.g. in chloroid plexus and kidney glomeruli, which is consistent with its role as a multifunctional regulator of endothelial cell growth and differentiation as disclosed by Breier et al. in Development (1992) 114:521-532. VEGF shares about 22% sequence identity, including a complete conservation of eight cysteine residues, according to Leung et al. in Science (1989) 246:1306-9, with human platelet-derived growth factor PDGF, a major growth factor for connective tissue. Alternatively spliced mRNAs have been identified for both VEGF and PDGF and these splicing products differ in their biological activity and receptor-binding specificity. VEGF is a potent vasoactive protein that has been detected in and purified from media conditioned by a number of cell lines including pituitary cells, such as bovine pituitary follicular cells (as disclosed by Ferrara et al. in Biochem. Biophys. Res. Comm. (1989) 161:851-858 and by Gospodarowicz et al. in Proc. Natl. Acad. Sci. USA (1989) 86: 7311-5), rat glioma cells (as disclosed by Conn. et al. in Proc. Natl. Acad. Sci. USA (1990) 87:1323-1327) and several tumor cell lines. Similarly, an endothelial growth factor isolated from mouse neuroblastoma cell line NB41 with an unreduced molecular mass of 43-51 kDa has been described by Levy et al. in Growth Factors (1989) 2:9-19.
VEGF was characterized as a glycosylated cationic 46 kDa dimer made up of two sub-units each with an apparent molecular mass of 23 kDa. It is inactivated by sulfhydryl reducing agents, resistant to acidic pH and to heating, and binds to immobilized heparin. VEGF has four different forms of 121, 165, 189 and 206 amino-acids due to alternative splicing of mRNA. The various VEGF species are encoded by the same gene. Analysis of genomic clones in the area of putative mRNA splicing also shows an intron/exon structure consistent with alternative splicing. The VEGF165 species is the molecular form predominantly found in normal cells and tissues. The VEGF 121 and VEGF165 species are soluble proteins and are capable of promoting angiogenesis, whereas the VEGF 189 and VEGF206 species are mostly cell-associated. All VEGF isoforms are biologically active, e.g. each of the species when applied intradermally is able to induce extravasation of Evans blue. However, VEGF isoforms have different biochemical properties which may possibly modulate the signalling properties of the growth factors. The VEGF 165, VEGF 189 and VEGF206 species contain eight additional cysteine residues within the carboxy-terminal region. The amino-terminal sequence of VEGF is preceded by 26 amino-acids corresponding to a typical signal sequence. The mature protein is generated directly following signal sequence cleavage without any intervening prosequence. Other VEGF polypeptides from the PDGF family of growth factors have been disclosed in U.S. Pat. No. 5,840,693. Purified and isolated VEGF-C cysteine deletion variants that bind to a VEGF tyrosine kinase receptor have been disclosed in U.S. Pat. No. 6,130,071.
Like other cytokines, VEGF can have diverse effects that depend on the specific biological context in which it is found. The expression of VEGF is high in vascularized tissues (e.g. lung, heart, placenta and solid tumors) and correlates with angiogenesis both temporally and spatially. VEGF has been shown to directly contribute to induction of angiogenesis in vivo by promoting endothelial cell growth during normal embryonic development wound healing, tissue regeneration and reorganization. Therefore VEGF has been proposed for use in promoting vascular tissue repair, as disclosed by EP-A-0,506,477. VEGF is also involved in pathological processes such as growth and metastasis of solid tumors and ischemia-induced retinal disorders such as disclosed in U.S. Pat. No. 6,114,320. VEGF expression is triggered by hypoxia so that endothelial cell proliferation and angiogenesis appear to be especially stimulated in ischemic areas. Finally, U.S. Pat. No. 6,040,157 discloses human VEGF2 polypeptides which have been putatively identified as novel vascular endothelial growth factors based on their amino-acid sequence homology to human VEGF. The latter document further discloses restoration of certain parameters in the ischemic limb by using a VEGF2 protein. However it is also known by Hariawala et al. in J. Surg. Res. (1996) 63(1):77-82 that a systemic administration of VEGF, in high doses over short periods of time, improves myocardial blood flow but produces hypotension in porcine hearts.
Placenta growth factor (hereinafter referred as PIGF) was disclosed by Maglione et al. in Proc. Natl. Acad. Sci. USA (1991) 88(20):9267-71 as a protein related to the vascular permeability factor. U.S. Pat. No. 5,919,899 discloses nucleotide sequences coding for a protein, named PIGF, which can be used in the treatment of inflammatory diseases and in the treatment of wounds or tissues after surgical operations, transplantations, burns of ulcers and so on. Soluble non-heparin binding and heparin binding forms, built up of 131 and 152 amino-acids respectively, have been described for PIGF which is expressed in placenta, trophoblastic tumors and cultured human endothelial cells, according to U.S. Pat. No. 5,776,755.
One problem to be solved by the present invention is to provide pharmaceutical compositions and methods for improving perfusion of the penumbra in the brain or perfusion of the jeopardized myocardium of patients suffering ischemic events, which will prove to be useful for the prevention and treatment of strokes and ischemic diseases, in particular ischemic cerebral infarction, acute myocardial infarction and chronic heart disease. Another problem to be solved by the present invention is to provide pharmaceutical compositions and methods for reducing or suppressing infarct expansion of the penumbra during ischemic cerebral infarction, making them useful for preventing and treating such a disease. Another problem to be solved by the present invention is to provide pharmaceutical compositions and methods for enhancing revascularization of acute myocardial infarcts, making them useful for preventing and treating such event. Another problem to be solved by the present invention is to provide a safe and effective route of administration of pharmaceutical compositions capable, namely with respect to the penumbra in the brain or the myocardium, of improving perfusion or reducing or suppressing infarct expansion or otherwise enhance revascularization of infarcts. Yet another problem to be solved by the present invention is to provide an effective means for the prevention and treatment of strokes and ischemic diseases, in particular ischemic cerebral infarction, acute myocardial infarction and chronic heart disease, which is devoid of adverse side-effects such as hypotension.